PROJECT SUMMARY Purinergic (P2X) receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, the molecular mechanisms for P2X receptor antagonism are unclear, especially for non-competitive antagonists where almost nothing is known. The study of P2X receptors has been handicapped by a paucity of small molecules that serve as selective high-affinity agonists and antagonists against the various receptor subtypes. Very recently, the applicant published atomic resolution structures of human P2X3 (hP2X3) in an apo/resting state, an agonist-bound/open-pore state, an agonist- bound/closed-pore/desensitized state and two competitive antagonist-bound states, identifying a novel cytoplasmic domain that he hypothesizes plays a pivotal and unique structural role in receptor activation and desensitization. The aims of this grant are designed to build off that work in order to firmly establish the principles of P2X receptor antagonism, determine the structural role of key cytoplasmic residues involved in P2X receptor activation and desensitization, and identify novel small molecule competitive and non-competitive antagonists directed at homo-trimeric P2X1 and P2X3 receptor subtypes and hetero-trimeric P2X2,3 receptors, utilizing ligand binding assays and electrophysiology to study function and X-ray crystallography and cryo electron microscopy to study structure. This research will be carried out by an applicant with excellent training in membrane protein biochemistry and a strong publication record. The training for this proposal will occur at the Vollum Institute of Oregon Health and Science University under the mentorship of Dr. Eric Gouaux, a world leader in the structural biology of ion channels and transporters who has previously transitioned numerous post-doctoral trainees to independence. To prepare the applicant for a successful transition to independence, Dr. Gouaux will oversee the candidate's structural biology training in cryo electron microscopy and continued growth in X-ray crystallography. In addition to Dr. Gouaux, an institute of established investigators supports the applicant with expertise in membrane protein biochemistry, ligand-receptor interactions/binding assays, and the electrophysiology of ion channels. This training will be essential for the candidate to become an independent investigator focused on studying the structure and function of purinergic receptors in order to develop pharmacological agents for the treatment of cardiovascular conditions such as angina, hypertension, and platelet aggregation.